Sir2a-based therapeutic and prophylactic methods

ABSTRACT

This invention provides methods for treating and for inhibiting the onset of cancer in a subject comprising administering an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis. This invention also provides a related method for inducing the death of a cell. This invention further provides a method for decreasing the amount of damage to a subject&#39;s cells caused by physical stress comprising administering agent that increases the amount of Sir2α in the subject&#39;s cells and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the subject&#39;s cells. This invention further provides related methods for prolonging the life-span of a subject, decreasing the amount of damage to a cell caused by physical stress, and prolonging the life-span of a cell. Finally, this invention provides two articles of manufacture for performing the instant methods.

This application claims the benefit of U.S. Provisional Application No.60/298,506, filed Jun. 15, 2001, the contents of which are herebyincorporated by reference into this application.

BACKGROUND OF THE INVENTION

The p53 tumor suppressor exerts anti-proliferative effects, includinggrowth arrest, apoptosis, and cell senescence, in response to varioustypes of stress (Levine, 1997; Prives and Hall, 1999; Vogelstein et al.,2000). Mutations within the p53 gene have been well documented in morethan half of all human tumors (Hollstein et al., 1994). Accumulatingevidence further indicates that, in the cells that retain wild-type p53,other defects in the p53 pathway also play an important role intumorigenesis (Prives and Hall, 1999; Lohrum and Vousden, 1999). Themolecular function of p53 that is required for tumor suppressioninvolves its ability to act as a transcriptional factor in regulatingdownstream target gene expression (reviewed in Nakano and Vousden, 2001;Yu et al., 2001).

p53 is a short-lived protein whose activity is maintained at low levelsin normal cells. Tight regulation of p53 is essential for its effect ontumorigenesis as well as maintaining normal cell growth. The precisemechanism by which p53 is activated by cellular stress is not completelyunderstood; it is generally thought to involve mainly post-translationalmodifications of p53, including phosphorylation and acetylation(reviewed in Appella and Anderson, 2000). Early studies demonstratedthat CBP/p300, a histone acetyl-transferase (HAT), acts as a coactivatorof p53 and potentiates its transcriptional activity as well asbiological function in vivo (Gu et al., 1997; Lill et al., 1997;Avantaggiati et al., 1997). Significantly, the observation of functionalsynergism between p53 and CBP/p300 together with its intrinsic HATactivity led to the discovery of a novel FAT (transcriptional factoracetyl-transferase) activity of CBP/p300 on p53. This finding alsoindicates that acetylation may represent a general functionalmodification for non-histone proteins in vivo (Gu and Roeder, 1997).

Through the use of site-specific acetylated p53 antibodies, CBP/p300mediated acetylation of p53 was further confirmed in vivo by a number ofstudies (Sakaguchi et al., 1998; Liu et al., 1999; Luo et al., 2000;Kobet et al., 2000; Ito et al., 2001). Significantly, the steady-statelevels of acetylated p53 are stimulated in response to various types ofstress, indicating the important role of p53 acetylation in stressresponse (reviewed in Ito et al., 2001).

By introducing a transcriptionally defective p53 mutant (p53^(Q25S26))into mice, it was found that the mutant mouse thymocytes and ES cellsfailed in undergoing DNA damage-induced apoptosis (Chao et al., 2000;Jimenez et al., 2000). Interestingly, this mutant protein wasphosphorylated normally at the N-terminus in response to DNA damage butcould not be acetylated at the C-terminus (Chao et al., 2000),supporting a critical role of p53 acetylation in p53-dependent apoptoticresponse (Chao et al., 2000; Luo et al., 2000).

Furthermore, it has been found that oncogenic Ras as well as PML canupregulate the levels of acetylated p53 in normal primary fibroblasts,and also induce premature senescence in a p53-dependent manner (Pearsonet al., 2000, Ferbeyre et al., 2000). p53 acetylation may also play acritical role in protein stabilization (Rodriguez et al., 2000; Nakamuraet al., 2000; Ito et al., 2001). In addition, another independent studyshowed that acetylation, but not phosphorylation of the p53 C-terminus,may be required to induce metaphase chromosome fragility in the cell (Yuet al., 2000).

In contrast, much less is known about the role of deacetylation inmodulating p53 function. The acetylation level of p53 is enhanced whenthe cells are treated with histone deacetylase (HDAC) inhibitors such asTrichostatin A (TSA). This observation led to the identification of aHDAC1 complex which is directly involved in p53 deacetylation andfunctional regulation (Luo et al., 2000; Juan et al., 2000).

PID/MTA2, a metastasis-associated protein 2, acts as an adaptor proteinto enhance HDAC1-mediated deacetylation of p53, but this activity can becompletely repressed by TSA (Luo et al., 2000). In addition, Mdm2, anegative regulator of p53, actively suppresses CBP/p300-mediated p53acetylation, and this inhibitory effect can be abrogated by tumorsuppressor p19^(ARF), suggesting that regulation of acetylation alsoplays a critical role in the p53-MDM2-p19^(ARF) feed back loop (Ito etal., 2001; Kobet et al., 2000).

The yeast silent information regulator 2 (Sir2) protein belongs to anovel family of histone deacetylases (reviewed in Guarente, 2000; Shore,2000). Sir2 activity is nicotinamide adenine dinucleotide(NAD)-dependent, but can not be inhibited by TSA (Imai et al., 2000;Landry et al., 2000a; Smith et al., 2000). The NAD-dependent deacetylaseactivity of Sir2 is essential for its functions, and this activity alsoconnects its biological role with cellular metabolism in yeast(Guarente, 2000; Imai et al., 2000; Lin et al., 2000; Smith et al.,2000). Recently, mammalian Sir2 homologs have been found to also containthe NAD-dependent histone deacetylase activity (Imai et al., 2000; Smithet al., 2000), further supporting the notion that the enzymatic activityis key to elucidating the molecular mechanism for its mediated functions(Min et al., 2001; Finnin et al., 2001).

Among Sir2 and its homolog proteins (HSTs) in yeast, Sir2 is the onlyprotein exclusively localized in nuclei, whose activity is critical forboth gene silencing and extension of yeast life-span (reviewed inGuarente, 2000). Based on protein sequence homology analysis, mouseSir2α and its human ortholog SIRT1 (or human Sir2α) are the closesthomologs to yeast Sir2 (Imai et al., 2000; Frye, 1999, 2000). However,their biological functions remain unclear.

SUMMARY OF THE INVENTION

This invention provides a method for treating a subject afflicted withcancer comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits the ability of Sir2α toinhibit p53-dependent apoptosis, thereby treating the subject.

This invention also provides a method for inhibiting the onset of cancerin a subject comprising administering to the subject a prophylacticallyeffective amount of an agent that inhibits the ability of Sir2α toinhibit p53-dependent apoptosis, thereby inhibiting the onset of cancerin the subject.

This invention further provides a method for inducing the death of acell comprising contacting the cell with an agent that inhibits theability of Sir2α to inhibit p53-dependent apoptosis, thereby inducingthe death of the cell.

This invention further provides a method for decreasing the amount ofdamage to a subject's cells caused by physical stress comprisingadministering to the subject a prophylactically effective amount of anagent that increases the amount of Sir2α in the subject's cells and/orthe ability of Sir2α to inhibit p53-dependent apoptosis in the subject'scells, thereby decreasing the amount of damage to the subject's cells.

This invention further provides a method for prolonging the life-span ofa subject comprising administering to the subject a prophylacticallyeffective amount of an agent that increases the amount of Sir2α in thesubject's cells and/or the ability of Sir2α to inhibit p53-dependentapoptosis in the subject's cells, thereby prolonging the subject'slife-span.

This invention further provides a method for decreasing the amount ofdamage to a cell caused by physical stress comprising contacting thecell with an agent that increases the amount of Sir2α in the cell and/orthe ability of Sir2α to inhibit p53-dependent apoptosis in the cell,thereby decreasing the amount of damage to the cell.

This invention still further provides a method for prolonging thelife-span of a cell comprising contacting the cell with an agent thatincreases the amount of Sir2α in the cell and/or the ability of Sir2α toinhibit p53-dependent apoptosis in the cell, thereby prolonging thelife-span of the cell.

Finally, this invention provides two articles of manufacture. The firstarticle of manufacture comprises a packaging material and an agentcontained therein that inhibits the ability of Sir2α to inhibitp53-dependent apoptosis, and a label indicating that the agent is usedfor treating a subject afflicted with cancer, inhibiting the onset ofcancer in a subject, and/or inducing the death of a cell.

The second article of manufacture comprises a packaging material and anagent contained therein that increases the amount of Sir2α in a celland/or the ability of Sir2α to inhibit p53-dependent apoptosis in acell, and a label indicating that the agent is used for decreasing theamount of damage to a subject's cells caused by physical stress,prolonging the life-span of a subject, decreasing the amount of damageto a cell caused by physical stress, and/or prolonging the life-span ofa cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Interactions between p53 and mammalian Sir2α both in vitro andin vivo. (A) Schematic representation of the high homology regionsbetween mouse Sir2α and human SIRT1 (hSIRT1). The core domain representsthe very conserved enzymatic domain among all Sir2 family proteins(Frye, 1999, 2000). (B) The interaction between p53 and hSIRT1 in H460cells. (C) The interaction between p53 and Sir2α in F9 cells. (D) Theinteraction between p53 and hSIRT1 in HCT116 cells either at the normalcondition (lanes, 1-3) or after DNA damage treatment by etoposide(lanes, 4-6). Western blot analyses of the indicated whole cell extract(WCE) (lanes 1, 4), or immunoprecipitates with anti-Sir2α antibody(IP/anti-Sir2α) (lanes 2, 5) prepared from different cell extracts, orcontrol immunoprecipitates with pre-immunoserum from the same extracts(lanes 3, 6), with anti-p53 monoclonal antibodies (DO-1 for human p53,421 for mouse p53), or anti-sir2a antibody. (E) Direct interactions ofSir2α with GST-p53. The GST-p53 full length protein (GST-p53) (lane 1),the N-terminus of p53 protein (1-73) (lane 2), the middle part of p53(100-290) (lane 3), the C-terminus of p53 (290-393) (lane 4), and GSTalone (lane 6) were used in GST pull-down assay with in vitro translated³⁵S-labeled full length mouse Sir2α.

FIG. 2. TSA-insensitive deacetylation of p53 by mammalian Sir2α. (A)Colloidal blue staining of a SDS-PAGE gel containing protein Marker(lane 1), a control eluate from M2 loaded with untransfected cellextract (lane 2), and 100 ng of the highly purified Flag-tagged Sir2αrecombinant protein (lane 3). (B) Deacetylation of p53 by Sir2α. 2.5 μgof ¹⁴C-labeled acetylated p53 (lane 1) was incubated with either thecontrol eluate (lane 4), the purified 10 ng of Sir2α (lanes 2 and 3), orthe same amount of Sir2α in the presence of 500 nM TSA (lane 5) for 60min at 30° C. NAD (50 μM) was also added in each reaction except lane 2.The proteins were analyzed by resolution on SDS-PAGE and autoradiography(upper) or Coomassie blue staining (lower). (C) Reduction of thesteady-state levels of acetylated p53 by both mouse Sir2α and humanSIRT1 expression. Western blot analysis of H1299 cell extracts from thecells cotransfected with p53 and p300 (lane 1), or in combination withSir2α (lane 2), or in combination with hSIRT1 (lane 4), or Sir2α-355A(lane 3), or hSIRT1-363Y (lane 5), or hSIRT5 (lane 6), or PARP (lane 7)by acetylated p53-specific antibody (upper) or DO-1 for total p53(lower). The highly conserved histidine residue at the core domain wasreplaced by alanine for mouse Sir2α (a.a. 355) Sir2α-355A), or replacedby tyrosine for human SIRT1 (a.a. 363) (hSIRT1-363Y). (D) Deacetylationof p53 by Sir2α in the presence of TSA. The acetylated p53 levels in thecells cotransfected with p53 and p300 (lanes 1, 3), or cotransfectedwith p53, p300 and Sir2α (lanes 2, 4). Cells were either not treated(lanes 1, 2) or treated with 500 nM TSA (lanes 3, 4).

FIG. 3. Abrogation of mammalian Sir2α mediated deacetylation of p53 bynicotinamide. (A) Sir2α-mediated deacetylation of p53 is inhibited bynicotinamide. 2.5 μg of ¹⁴C-labeled acetylated p53 (lane 1) wasincubated with 10 ng of purified Sir2α and 50 μM NAD alone (lane 2), orin the presence of either 5 mM of nicotinamide (lane 3) or 3 mM of 3-AB(3-aminobenzamide) (lane 4) for 60 min at 30° C. The proteins wereanalyzed by resolution on SDS-PAGE and autoradiography (upper) orCoomassie blue staining (lower). (B) Enhancement of endogenous p53acetylation levels by nicotinamide. Western blot analysis of cellextracts from untreated H460 cells, or the cells treated with etoposidealone (lane 2), or in combination with nicotinamide (lane 3). (C) TheSir2α-mediated deacetylation of endogenous p53 was abrogated in thepresence of nicotinamide. Cell extracts from the mock-infected MEFp53(+/+) cells (lanes 1-2, 5-6), or the pBabe-Sir2α infected cells(lanes 3-4, 7-8), either untreated (lanes 1, 3, 5, 7), or treated withetoposide and TSA (lanes 2, 4), or in combination with nicotinamide(lanes 6, 8) for 6 hr were analyzed by western blot with acetylatedp53-specific antibody (upper) or DO-1 for total p53 (lower). (D)Synergistic induction of p53 acetylation levels by TSA and nicotinamideduring DNA damage response. Western blot analysis of cell extracts fromthe H460 cells treated with etoposide alone (lane 2), or in combinationwith TSA (lane 3), or TSA and nicotinamide (lane 4), or TSA and 3-AB(lane 5) for 6 hr by acetylated p53-specific antibody (upper) or DO-1for total p53 (lower). The cell extracts from untreated cells (lane 1),or treated with ALLN (50 μM) were also included (lane 6).

FIG. 4. Repression of p53-mediated transcriptional activation bymammalian Sir2α. (A) MEF (p53−/−) cells were transiently transfectedwith 10 ng of CMV-p53 alone, or in combination with indicated amount ofSir2α together with the PG13-Luc reporter construct by calcium phosphateprecipitation essentially as previously described (Luo et al., 2000).(B) Western blot analysis of indicated transfected H1299 cell extractswith anti-p53 (DO-1), anti-p21 (C-19) and anti-β-actin. (C), (D) MEF(p53−/−) cells were transiently transfected with 10 ng of CMV-p53 alone,or in combination with 5 μg of either CMV-Sir2α, or CMV-hSIRT1, orCMV-hSIRT5 (C), or CMV-Sir2α-355A as indicated (D) together with thePG13-Luc reporter construct. All transfections were done in duplicateand representative experiments depict the average of three experimentswith standard deviations indicated.

FIG. 5. Inhibition of p53-dependent apoptosis by Sir2α. (A), (B) H1299cells were transfected with p53 alone, or cotransfected with p53 andSir2α, or cotransfected with p53 and Sir2α-355A. After transfection, thecells were fixed, stained for p53 by FITC-conjugated α-p53 antibody,analyzed for apoptotic cells (subG1) according to DNA content (PIstaining). (C), (D) Mammalian Sir2α has no effect on the Fas mediatedapoptosis. Both mock infected cells and pBabe-Sir2α infected MEFp53(−/−) cells were either not treated (1 and 2) or treated with 100ng/ml Fas antibody in presence of actinomycine D (0.25 μg/ml) (3 and 4).The experiments were repeated more than three times and the resultsdepict the average of three experiments with standard deviationsindicated (B), (C).

FIG. 6. Inhibition of the p53-dependent apoptosis in response to stressby mammalian Sir2α. (A) Repression of the apoptotic response to DNAdamage by Sir2α. Both mock infected cells and pBabe-Sir2α infected MEFp53(+/+) cells were either not treated (1 and 2) or treated with 20 μMetoposide. The cells were analyzed for apoptotic cells (subG1) accordingto DNA content (PI staining). Similar results were obtained for threetimes, and the representative data depict the average of threeexperiments with standard deviations indicated (B). (C) Subcellularlocalization of p53 and Sir2α in the pBabe-Sir2α infected IMR-90 cells.p53 and Sir2α were detected with either α-p53 (DO-1) (visualized bygreen fluorescence from secondary antibody staining with anti-mouseIgG-FITC), or affinity purified α-Sir2α antibody (visualized by redfluorescence from secondary antibody staining with anti-rabbit IgGconjugated to Alexa 568). The cells were counterstained with DAPI tovisualize the nuclei as essentially described before (Guo et al., 2000).Cells were either not treated (I) or treated with 100 μM H₂O₂ (II, III,IV) for 24 hr. (D) Inhibition of the apoptotic response to oxidativestress by mammalian Sir2α. Both mock infected cells and pBabe-Sir2αinfected cells were either not treated (I and III) or treated with 200μM H₂O₂ (II and IV). 24 hr later, the cells were photographed under amicroscope.

FIG. 7. Expression of a Sir2α point mutant (hSIRT1-363Y) increases thesensitivity of the cells in response to stress. (A) The levels of p53are induced by DNA damage in both mock infected and pBabe-hSIRT1-363Yinfected cells. Western blot analysis of the cell extracts from bothtypes of cells by anti-Sir2 polyclonal antibody (upper) or anti-p53monoclonal antibody (DO-1) (lower). Cells were either not treated (lanes1, 2) or treated with 20 μM of epotoside (lanes 3, 4) for 6 hr. (B)Expression of the Sir2α mutant enhances the acetylated p53 levelsinduced by DNA damage. The cell extracts obtained from treated oruntreated cells were first immunoprecipitated with anti-acetylated p53antibody and the immunoprecipitates were analyzed by western blot withα-p53 (DO-1). (C) DNA damage induced expression of p21 and Bax in bothmock infected and pBabe-hSIRT1-363Y infected cells. Both types of cellswere γ-irradiated (3 or 6 Gy), 3 hr later, the cells were collected forwestern blot analysis for p53, p21, Bax and β-actin. (D), (E) Expressionof the Sir2α mutant increases the sensitivity of the cells instress-induced apoptotic response. Both mock infected cells andpBabe-hSIRT1-363Y infected cells were either not treated (I and III) ortreated with 50 μM H₂O₂ (II and IV) (E), or treated with differentconcentrations of adriamycin as indicated. 48 hr later, the cells werecollected for analysis (D).

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Anti-sense nucleic acid” shall mean any nucleic acid which, whenintroduced into a cell, specifically hybridizes to at least a portion ofan mRNA in the cell encoding a protein (“target protein”) whoseexpression is to be inhibited, and thereby inhibits the target protein'sexpression.

“Catalytic nucleic acid” shall mean a nucleic acid that specificallyrecognizes a distinct substrate and catalyzes the chemical modificationof this substrate.

“DNAzyme” shall mean a catalytic nucleic acid that is DNA or whosecatalytic component is DNA, and which specifically recognizes andcleaves a distinct target nucleic acid sequence, which can be either DNAor RNA. Each DNAzyme has a catalytic component (also referred to as a“catalytic domain”) and a target sequence-binding component consistingof two binding domains, one on either side of the catalytic domain.

“Inhibiting” the onset of a disorder shall mean either lessening thelikelihood of the disorder's onset, or preventing the onset of thedisorder entirely. In the preferred embodiment, inhibiting the onset ofa disorder means preventing its onset entirely.

“Nucleic acid” shall mean any nucleic acid molecule, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

“Ribozyme” shall mean a catalytic nucleic acid molecule which is RNA orwhose catalytic component is RNA, and which specifically recognizes andcleaves a distinct target nucleic acid sequence, which can be either DNAor RNA. Each ribozyme has a catalytic component (also referred to as a“catalytic domain”) and a target sequence-binding component consistingof two binding domains, one on either side of the catalytic domain.

“Subject” shall mean any animal, such as a human, non-human primate,mouse, rat, guinea pig or rabbit.

“Treating” a disorder shall mean slowing, stopping or reversing thedisorder's progression. In the preferred embodiment, treating a disordermeans reversing the disorder's progression, ideally to the point ofeliminating the disorder itself.

EMBODIMENTS OF THE INVENTION

This invention provides a method for treating a subject afflicted withcancer comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits the ability of Sir2α toinhibit p53-dependent apoptosis, thereby treating the subject.

This invention also provides a method for inhibiting the onset of cancerin a subject comprising administering to the subject a prophylacticallyeffective amount of an agent that inhibits the ability of Sir2α toinhibit p53-dependent apoptosis, thereby inhibiting the onset of cancerin the subject. Such inhibition can be caused, for example, by alteringthe behavior of existing Sir2α or by decreasing Sir2α expression (e.g.,via anti-Sir2α nucleic acids such as anti-sense and catalytic nucleicacids such as ribozymes and DNAzymes). Sir2α is exemplified by humanSir2α having GenBank accession number AF083106 and the mouse Sir2αhaving GenBank accession number AF214646.

In the preferred embodiment of these methods, the subject is a human.The agent used in the instant methods can be any agent that inhibits p53deacylation, such as vitamin B₃ or nicotinamide. In a furtherembodiment, the instant methods further comprise administering to thesubject an agent that enhances p53-dependent apoptosis via a mechanismother than Sir2α inhibition, such as Trichostatin A or Etoposide.

This invention further provides a method for inducing the death of acell comprising contacting the cell with an agent that inhibits theability of Sir2α to inhibit p53-dependent apoptosis, thereby inducingthe death of the cell.

In the preferred embodiment of this method, the cell is a human cell.The agent used in the instant methods can be any agent that inhibits p53deacylation, such as vitamin B₃ or nicotinamide. In a furtherembodiment, this method further comprises contacting the cell with anagent that enhances p53-dependent apoptosis via a mechanism other thanSir2α inhibition, such as Trichostatin A or Etoposide.

This invention further provides a method for decreasing the amount ofdamage to a subject's cells caused by physical stress comprisingadministering to the subject a prophylactically effective amount of anagent that increases the amount of Sir2α in the subject's cells and/orthe ability of Sir2α to inhibit p53-dependent apoptosis in the subject'scells, thereby decreasing the amount of damage to the subject's cells.

The damage to the subject's cells can be any type of cellular damageincluding, for example, DNA damage and membrane damage. Physical stressincludes, without limitation, ultraviolet radiation and oxidation. Inthis method, the agent can be administered prior to, concurrently withor subsequent to the occurrence of the physical stress.

This invention further provides a method for prolonging the life-span ofa subject comprising administering to the subject a prophylacticallyeffective amount of an agent that increases the amount of Sir2α in thesubject's cells and/or the ability of Sir2α to inhibit p53-dependentapoptosis in the subject's cells, thereby prolonging the subject'slife-span.

In the preferred embodiment of the instant methods, the subject ishuman. The agent that increases the amount of Sir2α and/or the abilityof Sir2α to inhibit p53-dependent apoptosis can be, for example, aSir2α-encoding nucleic acid.

This invention further provides a method for decreasing the amount ofdamage to a cell caused by physical stress comprising contacting thecell with an agent that increases the amount of Sir2α in the cell and/orthe ability of Sir2α to inhibit p53-dependent apoptosis in the cell,thereby decreasing the amount of damage to the cell.

The damage to the cell can be any type of cellular damage including, forexample, DNA damage and membrane damage. Physical stress includes,without limitation, ultraviolet radiation and oxidation. In this method,the agent can be contacted with the cell prior to, concurrently with orsubsequent to the occurrence of the physical stress.

This invention still further provides a method for prolonging thelife-span of a cell comprising contacting the cell with an agent thatincreases the amount of Sir2α in the cell and/or the ability of Sir2α toinhibit p53-dependent apoptosis in the cell, thereby prolonging thelife-span of the cell.

In the preferred embodiment of the instant methods, the cell is a humancell. The agent that increases the amount of Sir2α and/or the ability ofSir2α to inhibit p53-dependent apoptosis can be, for example, aSir2α-encoding nucleic acid.

In this invention, administering agents can be effected or performedusing any of the various methods and delivery systems known to thoseskilled in the art. The administering can be performed, for example,intravenously, orally, nasally, via implant, transmucosally,transdermally, intramuscularly, and subcutaneously. The followingdelivery systems, which employ a number of routinely used pharmaceuticalcarriers, are only representative of the many embodiments envisioned foradministering the instant compositions.

Injectable drug delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g., ethanol, propylene glycol andsucrose) and polymers (e.g., polycaprolactones and PLGA's). Implantablesystems include rods and discs, and can contain excipients such as PLGAand polycaprolactone.

Oral delivery systems include tablets and capsules. These can containexcipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories,pessaries, gels and creams, and can contain excipients such assolubilizers and enhancers (e.g., propylene glycol, bile salts and aminoacids), and other vehicles (e.g., polyethylene glycol, fatty acid estersand derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueousgels, creams, multiple emulsions, microemulsions, liposomes, ointments,aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon basesand powders, and can contain excipients such as solubilizers, permeationenhancers (e.g., fatty acids, fatty acid esters, fatty alcohols andamino acids), and hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone). In one embodiment, the pharmaceutically acceptablecarrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systemsinclude vehicles such as suspending agents (e.g., gums, xanthans,cellulosics and sugars), humectants (e.g., sorbitol), solubilizers(e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g.,sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservativesand antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid),anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

Finally, this invention provides two articles of manufacture. The firstarticle of manufacture comprises a packaging material and an agentcontained therein that inhibits the ability of Sir2α to inhibitp53-dependent apoptosis, and a label indicating that the agent is usedfor treating a subject afflicted with cancer, inhibiting the onset ofcancer in a subject, and/or inducing the death of a cell.

The second article of manufacture comprises a packaging material and anagent contained therein that increases the amount of Sir2α in a celland/or the ability of Sir2α to inhibit p53-dependent apoptosis in acell, and a label indicating that the agent is used for decreasing theamount of damage to a subject's cells caused by physical stress,prolonging the life-span of a subject, decreasing the amount of damageto a cell caused by physical stress, and/or prolonging the life-span ofa cell.

This invention is illustrated in the Experimental Details section thatfollows. This section is set forth to aid in an understanding of theinvention but is not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

Experimental Details Introduction

Since homologues of Sir2 have been identified in almost all organismsexamined including bacteria, which has no histone proteins (reviewed inGuarente, 2000; Frye, 1999; 2000), it is likely that Sir2 also targetsnon-histone proteins for functional regulation (Muth et al., 2001). Ourpreliminary results uncovered a novel activity in protein fractions frommammalian nuclear extract that could effectively deacetylate p53 in thepresence of TSA. In support of the notion that mammalian Sir2α targetsp53 for functional regulation, we present evidence that mouse Sir2α aswell as human SIRT1 can directly bind p53 both in vitro and in vivo, andpromotes cell survival under stress by specifically repressingp53-dependent apoptotic response.

Synopsis of Experimental Findings

The NAD-dependent histone deacetylation of Sir2 connects cellularmetabolism with gene silencing as well as aging in yeast. Here, we showthat mammalian Sir2α physically interacts with p53 and attenuatesp53-mediated functions. Nicotinamide (Vitamin B3) inhibits anNAD-dependent p53 deacetylation induced by Sir2α, and also enhances thep53 acetylation levels in vivo. Furthermore, Sir2α repressesp53-dependent apoptosis in response to DNA damage and oxidative stress,whereas expression of a Sir2α point-mutant increases the sensitivity ofcells in the stress response. Thus, our findings implicate a novel p53regulatory pathway mediated by mammalian Sir2α. These results havesignificant implications regarding an important role for Sir2α inmodulating the sensitivity of cells in p53-dependent apoptotic responseand the possible effect in cancer therapy.

A. Results

Mammalian Sir2α Interacts with P53 Both In Vitro and in Vivo

Since mouse Sir2α shares a highly conserved region at the C-terminuswith human SIRT1 (FIG. 1A), but not with any other mammalian Sir2homologs (Frye, 1999; 2000), we developed a polyclonal antibody againstthe C-terminus (amino acid 480-737) of mouse Sir2α. By western blotanalysis, this antibody can detect both mouse Sir2α and human SIRT1proteins, but not other human Sir2 homologs (see FIG. 1B,C). Next, weused this antibody to investigate whether p53 interacts with Sir2α orhSIRT1 in normal cells. Cell extracts from both human (H460) and mousecells (F9), which express wild-type p53 proteins, wereimmunoprecipitated with α-Sir2α, or with the pre-immune serum. Westernblot analysis revealed that this antibody immunoprecipitated both Sir2αand hSIRT1 (lower panels, FIG. 1B, 1C). More importantly, both human andmouse p53 were clearly detected in the respective α-Sir2αimmunoprecipitations from cell extracts, but not in the controlimmunoprecipitations with the preimmune serum, indicating that p53interacts with mammalian Sir2α in normal cells. Interestingly, thisinteraction was strongly detected in the cells after DNA damagetreatment (FIG. 1D), suggesting that the possible regulation of p53 bymammalian Sir2α may be still effective after DNA-damage.

Furthermore, we tested whether Sir2α directly interacts with p53 invitro. As shown in FIG. 1E, ³⁵S-labeled in vitro translated Sir2αstrongly bound to immobilized GST-p53 but not to immobilized GST alone(lane 1 vs 6). Moreover, Sir2α tightly bound to the C-terminal domain ofp53 (GST-p53CT) (lane 4, FIG. 1E), also bound to the central DNA-bindingdomain (GST-p53M), but showed no binding to the N-terminal domain of p53(GST-p53NT) (lane 3 vs 2, FIG. 1E). Thus, the above findings demonstratethat p53 interacts with mammalian Sir2α both in vitro and in vivo.

Deacetylation of p53 by Mammalian Sir2α

In order to test whether p53 could be specifically deacetylated bymammalian Sir2α in vitro, the mouse Sir2α protein was expressed with theN-terminal Flag epitope in cells and purified to near homogeneity on theM2-agarose affinity column (lane 3, FIG. 2A). As shown in FIG. 2B,¹⁴C-labeled acetylated p53 was efficiently deacetylated by purifiedSir2α (lane 3), but not by a control eluate (lane 4). Importantly, NADis required for Sir2α-mediated deacetylation of p53 (lane 2 vs. lane 3,FIG. 2B). In addition, the deacetylase inhibitor TSA, whichsignificantly abrogates HDAC1-mediated deacetylase activity on p53 (Luoet al., 2000), had no apparent effect on Sir2α-mediated p53deacetylation (lane 5, FIG. 2B). These results indicate that the Sir2αcan strongly deacetylate p53 in vitro, and that this activity depends onNAD.

To establish the role for mammalian Sir2α in deacetylating p53 in cells,we used an acetylated p53-specific antibody to monitor the steady-statelevels of acetylated p53 in vivo (Luo et al., 2000). As indicated inFIG. 2C, a high level of acetylated p53 was found in the cellscotransfected with p300 and p53 (lane 1); however, p53 acetylationlevels were significantly abolished by expression of either Sir2α orhSIRT1 (lanes 2, 4). In contrast, a point mutation at the highlyconserved histidine residue at the core domain (Sir2α{tilde over ()}355A and hSIRT1-363Y) effectively abolished the deacetylase activity(lane 3 vs. 2, lane 5 vs. 4, FIG. 2C). Furthermore, neither SIRT5,another human Sir2 homolog, nor poly(ADP-ribose) polymerase (PARP),whose activity is also NAD-dependent (reviewed in Vaziri et al., 1997),had any significant effect on p53 acetylation (lanes 6, 7, FIG. 2C). Inaddition, in contrast to HDAC-mediated deacetylation of p53 (Luo et al.,2000), Sir2α still strongly deacetylated p53 in the presence of TSA(lane 4 vs 3, FIG. 2D) even though the steady state level of acetylatedp53 was elevated when the cells were treated with TSA (lane 3 vs 1, FIG.2D). Taken together, these data implicate a strong TSA-independent p53deacetylation activity of mammalian Sir2α.

Inhibition of Sir2α-Mediated p53 Deacetylation by Nicotinamide

To further elucidate the in vivo effect by endogenous Sir2α, we tried toidentify an inhibitor for Sir2α-mediated deacetylase activity on p53.Deacetylation of acetyl-lysine by Sir2α is tightly coupled to NADhydrolysis, producing nicotinamide and a novel acetyl-ADP-ribosecompound (1-O-acetyl-ADPribose) (Landry et al., 2000b; Tanner et al.,2000; Tanny and Moazed, 2001). Although the molecular mechanism of Sir2mediated NAD-dependent deacetylation needs to be detailed, it wasproposed that formation of an enzyme-ADP-ribose intermediate through NADhydrolysis is critical for this chemical reaction (Landry et al.,2000b). Since nicotinamide is the first product from hydrolysis of thepyridinium-N-glycosidic bond of NAD, it may function as an inhibitor forits deacetylase activity (Landry et al., 2000b). We thus tested whethernicotinamide is able to inhibit the deacetylase activity of Sir2α onacetylated p53 in vitro.

Similar reactions as described above (FIG. 2B), were set up byincubating labeled-p53 substrate, recombinant Sir2α and NAD (50 μM)alone, or in combination with nicotinamide (5 mM). As shown in FIG. 3A,¹⁴C-labeled acetylated p53 was efficiently deacetylated by Sir2α (lane2), however, the deacetylation activity was completely inhibited in thepresence of nicotinamide (lane 3 vs 2). As a negative control, 3-AB(3-aminobenzamide), a strong inhibitor of PARP which is involved inanother type of NAD-dependent protein modifications (Vaziri et al.,1997), showed no significant effect on Sir2α mediated deacetylation(lane 4 vs. 3, FIG. 3A). Significantly, the cellular levels ofacetylated p53 induced by DNA damage were enhanced when the cells weretreated with nicotinamide (lane 3 vs. 2, FIG. 3B), indicating apotential effect of nicotinamide on endogenous Sir2α-mediated p53deacetylation.

Furthermore, we examined the effect of Sir2α expression on endogenouslevels of acetylated p53. Mouse embryonic fibroblast (MEF) cells, whichexpress the wild type of p53, were infected with either a pBaberetrovirus empty vector or a pBabe retrovirus containing Sir2α, andcultured for a week under pharmacological selection. We first examinedthe protein levels of p53 activation in response to DNA damage in thesecells by western blot analysis. Similar protein levels of p53 activationwere induced in both types of cells after etoposide treatment for 6 hrs(lanes 3, 4 vs. lanes 1, 2, lower panel, FIG. 3C). In the mock-infectedcells, as expected, the acetylation level of p53 was significantlyenhanced by DNA damage (lane 2 vs 1, Upper panel, FIG. 3C). However, thesame DNA damage treatment failed to stimulate the p53 acetylation in thepBabe-Sir2α infected cells even in the presence of TSA (lane 4 vs 2,Upper panel, FIG. 3C), indicating that Sir2α expression results indeacetylation of endogenous p53. Notably, this Sir2α-mediated effect wascompletely abrogated by nicotinamide treatment (lane 8 vs 6, FIG. 3C).Thus, these data indicate that Sir2α mediated deacetylation of p53 canbe inhibited by nicotinamide both in vitro and in vivo.

Maximum Induction of p53 Acetylation Levels in Normal Cells RequiresInhibition of Endogenous Sir2α Activity

After we found that nicotinamide has a strong inhibitory effect on Sir2αmediated deacetylation in vivo (FIG. 3C), we further test whether theendogenous Sir2α is critical in regulating the p53 acetylation levels innormal cells during the DNA damage response.

As indicated in FIG. 3D, after the wild-type p53 containing human lungcarcinoma cells (H460) were treated by etoposide, acetylation of p53 wasindeed induced (lane 2, vs. 1). In contrast, no significant p53acetylation was detected in the cells treated with a proteasomeinhibitor ALLN (Lane 6, FIG. 3D), indicating that the observedstimulation of p53 acetylation is induced by DNA damage, not through p53stabilization. We have previously shown that p53 can be deacetylated bya PID/MTA2/HDAC1 complex, whose activity is completely abrogated in thepresence of TSA (Luo et al., 2000). Therefore, the mild enhancement ofthe acetylation level of p53 by TSA during DNA damage response may bedue mainly to its inhibitory effect on endogenous HDAC1-mediateddeacetylase activity (lane 3 vs 2, FIG. 3D). Strikingly, a superinduction of p53 acetylation was shown when the cells were treated withboth TSA and nicotinamide (lane 4 vs. 3, FIG. 4D). In contrast, 3-ABtreatment had no effect on the level of p53 acetylation (lane 5 vs 3,FIG. 3D), indicating that PARP-mediated poly-ADP ribosylation has noeffect on p53 acetylation. Similar results were also observed in othercell types including either mouse cells (MEFs, F9) or human cells (BL2,HCT116). Thus, these data clearly indicate that maximum induction of p53acetylation requires inhibitors for both types of deacetylases (HDAC1and Sir2α), and that endogenous Sir2α plays an important role in theregulation of the p53 acetylation levels induced by DNA damage.

Repression of p53-Mediated Functions by Mammalian Sir2α Requires itsDeacetylase Activity

To determine the functional consequence of mammalian Sir2α-mediateddeacetylation of p53, we tested its effect on p53-mediatedtranscriptional activation. A mammalian p53 expression vector (CMV-p53),alone or in combination with different amounts of mouse Sir2α expressingvector (CMV-Sir2α) was cotransfected into MEF (p53^(−/−)) cells alongwith a reporter construct containing synthetic p53 binding sites placedupstream of the luciferase gene (PG13-Luc). As shown in FIG. 4A, Sir2αstrongly repressed p53-mediated transactivation in a dose-dependentmanner (up to 21 fold), and expression of human SIRT1 showed a similareffect on the p53 target promoter (FIG. 4C). Significantly, Sir2αexpression also attenuates p53-dependent induction of endogenous p21expression (lane 3 vs 2, FIG. 4B). Neither the Sir2α-355A mutant norSIRT5, both of which are defective in p53 deacetylation (FIG. 2C), hadany effect on the p53-mediated transactivation (FIG. 4C, D). These datasuggest that mammalian Sir2α specifically represses p53-dependenttransactivation, and that this repression requires its deacetylaseactivity.

To further test the biological role of mammalian Sir2α, we examined itsmodulation on p53-dependent apoptosis. p53 null cells (H1299) weretransfected with p53 alone or cotransfected with p53 and Sir2α. Thetransfected cells were fixed, stained for p53, and analyzed forapoptotic cells (SubG1) (Luo et al., 2000). As indicated in FIG. 5A,overexpression of p53 alone induced significant apoptosis (32.3% SubG1).However, co-transfection of p53 with Sir2α significantly reduced thelevel of apoptosis (16.4% SubG1), while the mutant Sir2α-355A wasseverely impaired in this effect (29.5% SubG1) (FIG. 5A, B). Takentogether, these data demonstrate that mammalian Sir2α is involved in theregulation of both p53-mediated transcriptional activation andp53-dependent apoptosis, and that the deacetylase activity is requiredfor these Sir2α-mediated effects on p53.

The Role of Mammalian Sir2α in Stress-Induced Apoptotic Response

Our data have indicated that mammalian Sir2α can deacetylate p53 both invitro and in vivo (FIG. 2). More importantly, Sir2α can attenuatep53-mediated transcriptional activation (FIG. 4). To elucidate thephysiological significance for this Sir2α-mediated regulation, weexamined its effect on DNA damage-induced apoptotic response. For thisstudy, we chose the same MEF (p53^(+/+)) cells as described above (FIG.3C), which were infected with either a pBabe retrovirus empty vector ora pBabe retrovirus containing Sir2α. After the DNA damage treatment byetoposide, the cells were stained with PI and analyzed by flowcytometric analysis for apoptotic cells (SubG1) according to DNAcontent. As shown in FIG. 6A, the cells mock infected with thepBabe-vector were susceptible to etoposide-induced cell death, withabout 48% of the cells apoptotic after exposure to 20 μM of etoposide (3vs. 1, FIG. 6A). In contrast, the pBabe-Sir2α-infected MEF (p53^(+/+))cells were more resistant to apoptosis induced by the same dose ofetoposide, with only 16.4% apoptotic cells (4 vs. 3, FIG. 6A, B). Sinceno significant apoptosis was detected in MEF (p53^(−/−)) cells by thesame treatment, the induced apoptosis observed in MEF (p53^(+/+)) cellsis totally p53-dependent. Thus, these results indicate that Sir2αsignificantly inhibits p53-dependent apoptosis in response to DNAdamage.

Since stimulation of p53 acetylation as well as p53-dependent apoptosishave also been implicated in many other types of stress response(reviewed in Ito et al., 2001), we examined the role of mammalian Sir2αin the oxidative stress response. Recent studies have indicated thatoxidative stress-induced cell death is p53-dependent (Yin et al., 1998;Migliaccio et al., 1999). We chose early-passage normal human fibroblast(NHF) IMR-90 cells for this study since it has been demonstrated thatp53-dependent apoptosis can be strongly induced by hydrogen peroxidetreatment in these cells (Chen et al., 2000). IMR-90 cells were infectedwith either a pBabe retrovirus empty vector or a pBabe retroviruscontaining Sir2α, and cultured for a week under pharmacologicalselection. By immunofluorescence staining, we found that p53 in theseinfected cells was induced significantly after hydrogen peroxidetreatment, along with Sir2α localized in the nuclei detected byimmunostaining with specific antibodies (FIG. 6C). Importantly, Sir2αexpression significantly promotes cell survival under oxidative stress.As indicated in FIG. 6D, the cells mock infected with the pBabe-vectorwere susceptible to H₂O₂-induced cell death, with more than 80% of thecells being killed after 24 hr exposure to 200 μM H₂O₂ (II vs. I). Incontrast, the pBabe-Sir2α infected cells were much more resistant todeath by the same dose of H₂O₂, with about 70% of the cells survivingafter 24 hr of H₂O₂ treatment (IV vs. III, FIG. 6D).

Taken together, these results suggest that mammalian Sir2α promotes cellsurvival under stress by inhibiting p53-dependent apoptosis.

Mammalian Sir2α has No Effect on p53-Independent Cell Death Induced byAnti-Fas.

In order to determine the specificity of mammalian Sir2α-mediatedprotection of cells from apoptosis, we examined whether Sir2α has anyeffect on p53-independent, Fas-mediated apoptosis. The MEF (p53^(−/−))cells were first infected with either a pBabe retrovirus empty vector ora pBabe retrovirus containing Sir2α, then cultured for a week underpharmacological selection. After the treatment by anti-Fas (100 ng/ml)for 24 hrs, the cells were harvested and further analyzed for apoptoticcells (SubG1). As shown in FIG. 5D, the cells mock infected with thepBabe vector were susceptible to anti-Fas induced cell death, with about31.7% of the cells becoming apoptotic. However, in contrast to thestrong protection of p53-dependent apoptosis by Sir2α during DNA damageresponse in the MEF (p53^(+/+)) cells (FIG. 6A, B), Sir2α expression hadno significant effect on Fas-mediated apoptosis in the MEF (p53^(−/−))cells (FIG. 5C, D). Thus, these results further support a specific rolefor mammalian Sir2α in regulating p53-mediated apoptosis.

Expression of a Sir2α Point-Mutant Increases the Sensitivity of Cells inthe Stress Response

To further demonstrate that endogenous Sir2α regulates endogenous p53under normal conditions, we introduced a Sir2α point-mutant(hSIRT1-363Y), which is functionally-defective in p53 deacetylation(FIG. 2C), into normal human cells. IMR-90 cells were infected witheither a pBabe retrovirus empty vector or a pBabe retrovirus containinghSIRT1-363Y, and cultured for a week under pharmacological selection. Asindicated in FIG. 7A, the pBabe-hSIRT1-363Y infected cells expressed asignificant amount of the mutant protein whereas similar levels of p53were induced in both types of cells after DNA damage treatment (lanes 3,4 vs. lanes 1, 2, FIG. 7A). Significantly, the levels of acetylated p53were strongly enhanced in the pBabe-hSIRT1-363Y-infected cells,indicating that hSIRT1-363Y functions as a dominant negative mutant andinhibits endogenous Sir2α-mediated deacetylation of p53 (lane 4 vs. 3,FIG. 7B). We further test whether hSIRT1-363Y expression has any effecton p53 target genes induced by DNA damage. As indicated in FIG. 7C, bothp21 and Bax were induced in the cells after DNA damage, interestinglyhowever, the expression levels of both p21 and Bax in hSIRT1-363Yinfected cells were significantly higher than those in mock-infectedcells, indicated that hSIRT1-363Y expression abrogates the endogenousSir2α-mediated repression on p53-dependent transactivation. Moreover,although the IMR-90 cells were susceptible to H₂O₂-induced cell deathafter exposure to 200 μM H₂O₂ (FIG. 6D), the cells were relativelyresistant to the treatment with a lower concentration of H₂O₂ (50 μM)(II vs. I, FIG. 7E). In contrast, hSIRT1-363Y expression led to thecells very sensitive to such a mild treatment (50 μM H₂O₂), with lessthan 30% of the cells surviving (IV vs. III, FIG. 7E). In order tocollaborate these results, we also test whether hSIRT1-363Y expressionincreases the sensitivity of the cells in DNA damage-induced cell death.As shown in FIG. 7D, the pBabe-hSIRT1-363Y infected cells are much moresensitive to DNA damage-induced cell death when the cells were treatedwith different concentrations of a DNA damage-induced reagentadriamycin.

Taken together, these results suggest that endogenous Sir2α iscritically involved in deacetylating p53 as well as regulatingp53-mediated biological functions under physiological conditions.

B. Discussion

The present data reveal the existence of a novel p53 regulatory pathwaythat is controlled by mammalian Sir2α. Sir2α is involved in genesilencing and extension of life span in yeast and C. elegans (reviewedin Guarente, 2000; Shore, 2000; Kaeberlein et al., 1999; Tissenbaun andGuarente, 2001). Although the mammalian homolog has also been shown tocontain a unique histone deacetylase activity (Imai et al., 2000; Smithet al., 2000), its biological function remains to be elucidated. We showhere (i) that p53 strongly binds to mouse Sir2α as well as its humanortholog hSIRT1 both in vitro and in vivo, (ii) that p53 is a substratefor the NAD-dependent deacetylase of mammalian Sir2α, (iii) that theSir2α-mediated deacetylation antagonizes p53-dependent transcriptionalactivation and apoptosis, (iv) that the Sir2α-mediated deacetylation ofp53 is inhibited by nicotinamide both in vitro and in vivo, (v) thatSir2α specifically inhibits p53-dependent apoptosis in response to DNAdamage as well as oxidative stress, but not the p53-independent,Fas-mediated cell death, and (vi) expression of a Sir2α point mutantincreases the sensitivity of the cells in response to stress. Theseresults are especially relevant to the multiple regulatory pathways ofp53 in vivo and, since the acetylation levels of p53 are stimulated inresponse to various types of stress, to the role of mammalian Sir2α instress response.

The Sir2α-Mediated Pathway is Critical for Cells Under Stress

Our study implicates a novel, Mdm2-independent, negative regulatorypathway for p53, which further supports the views that there aremultiple pathways in cells for tight regulation of p53 function (Privesand Hall, 1999; Appella and Anderson, 2000). In normal cells, Mdm2 isthe major negative regulator for p53, and Mdm2-mediated repressionappears sufficient to downregulate p53 activity. Interestingly, while noobvious effect by Sir2α expression was observed in cells at normalconditions, Sir2α becomes critical in protecting cells from apoptosiswhen cells were either treated by DNA damage or under oxidative stress(FIG. 6). Therefore, we propose that this Sir2α-mediated pathway iscritical for cell survival when the p53 negative control mediated byMdm2 is severely attenuated in response to DNA damage or other types ofstress.

In this regard, p53 is often found as latent forms and the levels of p53protein are very low in unstressed cells, mainly due to the tightregulation by Mdm2 through functional inhibition and protein degradationmechanisms (reviewed in Freedman et al., 1999). However, in response toDNA damage, p53 is phosphorylated at multiple sites at the N-terminus.These phosphorylation events contribute to p53 stabilization andactivation by preventing binding with Mdm2 (reviewed in Appella andAnderson, 2000; Shieh et al., 1997). Mdm2 itself is also phosphorylatedby ATM during DNA damage response, and this modification attenuates itsinhibitory potential on p53 (Maya et al., 2001). Furthermore, while p53is strongly stabilized and highly acetylated in stressed cells,acetylation of the C-terminal multiple lysine sites may occupy the samesites responsible for Mdm2-mediated ubiquitination (Rodriguez et al.,2000; Nakamura et al., 2000), and the highly acetylated p53 can not beeffectively degraded by Mdm2 without deacetylation (Ito et al., 2001).Thus, in contrast to unstressed cells, the main p53 negative regulatorypathway mediated by Mdm2 is severely blocked at several levels inresponse to DNA damage (Maya et al., 2001). Under these circumstances,the Sir2α-mediated regulation may become a major factor in controllingp53 activity, making it possible for cells to adjust the p53 activityfor DNA repair before committing to apoptosis.

Attenuation of p53-Mediated Transactivation by Sir2α

Earlier studies indicated that p53-mediated transcriptional activationis sufficient and also absolutely required for its effect on cell growtharrest, while both transactivation-dependent and -independent pathwaysare involved in p53-mediated apoptosis (reviewed in Prives and Hall,1999). However, there is now growing evidence showing that p53 caneffectively induce apoptosis by activating pro-apoptotic genes in vivo(reviewed in Nakano and Vousden, 2001; Yu et al., 2001). Thus, tightregulation of p53-mediated transactivation is critical for its effect onboth cell growth and apoptosis (Chao et al., 2000; Jimenez et al.,2000).

Recent studies indicate that the intrinsic histone deacetylase activityof Sir2α is essential for its mediated functions (reviewed in Gurante,2000). Reversible acetylation was originally identified in histones,however, accumulating evidence indicates that transcriptional factorsare also functional targets of acetylation (reviewed in Sterner andBerger, 2000; Kouzarides, 2000). Thus, the transcriptional attenuationmediated by histone deacetylases may act through the effects on bothhistone and non-histone transcriptional factors (Sterner and Berger,2000; Kuo and Allis, 1998). Interestingly, microarray surveys fortranscriptional effects of Sir2 in yeast revealed that Sir2 appears torepress amino acid biosynthesis genes, which are not located attraditional “silenced” loci (Bernstein et al., 2000). Thus, in additionto silencing (repression) at telomeres, mating type loci and ribosomalDNA (reviewed in Guarente, 2000; Shore, 2000), Sir2 may also be targetedto specific endogenous genes for transcriptional regulation in yeast.

In fact, there are at least seven different Sir2 homologs present inmammalian cells, but only mouse Sir2α and human SIRT1 are trulyorthologs to yeast Sir2 based on the amino acid sequence homology andprotein structure similarity (Frye, 1999, 2000; Imai et al., 2000). Inaddition, mouse SIR2L2 and SIR2L3 (or Human SIRT2 and SIRT3), arecytoplasmic proteins (Yang et al., 2000; Perrod et al., 2001). We havefound that neither human SIRT5 nor SIRT6 binds to, or has any effect indeacetylating p53 in vivo (FIG. 2C), further supporting the specificityof the regulation of p53 by mammalian Sir2α. Furthermore, in contrast tothe yeast counterpart Sir2, the mouse Sir2α protein does not co-localizewith nucleoli, telomeres or centromeres by co-immunofluorescence assay,indicating that this protein is not associated with the most highlytandemly repeated DNA in the mouse genome. The immunostaining pattern ofSir2α indicates that mammalian Sir2α is, similar to HDAC1, broadlylocalized in the nucleus, further supporting the notion that mammalianSir2α may be recruited to specific target genes for transcriptionalregulation in vivo.

Our results suggest that mammalian Sir2α inhibits p53-mediated apoptosisthrough attenuation of the transcriptional activation potential of p53.Our study also predicts that other cellular factors may use a similarmechanism to recruit Sir2 family proteins for TSA-insensitivetranscriptional regulation in mammalian cells.

Novel Implications for Cancer Therapy

Inactivation of p53 functions has been well documented as a commonmechanism for tumorigenesis (Vogelstein et al., 2000). Many cancertherapy drugs have been designed based on either reactivating p53functions or inactivating p53 negative regulators. Since p53 is stronglyactivated in response to DNA damage mainly through attenuation of theMdm2-mediated negative regulatory pathway (Maya et al., 2001), many DNAdamage-inducing drugs such as etoposide are very effective antitumordrugs in cancer therapy (reviewed in Chresta and Hickman, 1996; Lutzkerand Levine, 1996). Based on our results that the maximum induction ofp53 acetylation in normal cells requires both types of deacetylaseinhibitors in addition to DNA damage, there are at least three differentp53-negative regulatory pathways in mammalian cells. Interestingly,inhibitors for HDAC-mediated deacetylases, including sodium butyrate,TSA, SAHA and others, have been also proposed as antitumor drugs(reviewed in Marks, et al., 2001; Butler et al., 2000; Yoshida et al.,1995). Thus, we envision as one embodiment of this invention proposethat the combining DNA damage drugs, HDAC-mediated deacetylaseinhibitors, and Sir2α-medicated deacetylase inhibitors, in cancertherapy for maximally activating p53.

In contrast to PID/HDAC1-mediated p53 regulation (Luo et al., 2000), ourresults have shown that mammalian Sir2α-mediated effect on p53 isNAD-dependent, indicating that this type of regulation is closely linkedto cellular metabolism (reviewed in Guarente 2000; Campisi, 2000). Infact, null mutants of NPT1, a gene that functions in NAD synthesis, showphenotypes similar to that of Sir2 mutants in silencing (Smith et al.,2000) and in life extension in response to caloric restriction in yeast(Lin et al., 2000). Thus, metabolic rate may play a role inSir2α-mediated regulation of p53 function and, perhaps, modulate thesensitivity of cells in p53-dependent apoptotic response.

C. Experimental Procedures

Plasmids and Antibodies

To construct Sir2α expression constructs, the full-length cDNA wassubcloned from pET28a-Sir2α (Imai et al., 2000) into pcDNA3 or pBabepurovector. Site-directed mutation was generated in the plasmid pRS305—Sir2αusing the Gene Edit system (Promega). To construct the human SIRT1expression construct, DNA sequences corresponding to the full-lengthhSIRT1 (Frye, 1999) were amplified by PCR from Marathon-Ready Hela cDNA(Clontech), and initially subcloned into pcDNA3.1/V5-His-Topo vector(Invitrogen), and then subcloned with a Flag-tag into a pCIN4 vector forexpression (Gu et al., 1999). To prepare the Sir2α antibody that canrecognize both human and mouse Sir2α, we made a polyclonal antibodyagainst the highly conserved C-terminus of Sir2α. DNA sequencescorresponding to this region (480-737) were amplified by PCR andsubcloned into pGEX-2T (Pharmacia). α-Sir2α antisera were raised inrabbits against the purified GST-Sir2α(480-737) fusion protein(Covance), and further affinity-purified on both protein-A and antigencolumns. By western blot analysis and immunofluorescent staining, thisantibody can detect both mouse Sir2α and human SIRT1 proteins.

In Vitro p53 Deacetylation Assay

The Flag-tagged Sir2α cells were established and expanded in DMEMmedium, and cell extracts were prepared essentially as previouslydescribed (Luo et al., 2000; Gu et al., 1999). The proteins werepurified under a very high stringency condition (300 mM NaCl and 0.5%NP-40). The eluted proteins were resolved by a SDS-PAGE gel and analyzedby colloidal blue staining (Novex). Acetylated GST-p53 was prepared byp53 acetylation assay as previously described (Gu and Roeder, 1997) andfurther purified on glutathione-Sepharose (Luo et al., 2000). The¹⁴C-labeled acetylated p53 (2.5 μg) was incubated with purified Sir2α(10 ng) at 30° C. for 1 hr either in the presence of 50 μM NAD or asindicated. The reactions were performed in a buffer containing 50 mMTris-HCl (pH 9.0), 50 mM NaCl, 4 mM MgCl₂, 0.5 mM DTT, 0.2 mM PMSF,0.02% NP-40 and 5% glycerol. The reactions were resolved on SDS-PAGE andanalyzed by Coomassie blue staining and autoradiography.

Virus Infection and Stress Response

All MEF cells were maintained in DMEM medium supplemented with 10% fetalbovine serum, and the IMR-90 cells were maintained in Eagle's minimalessential medium supplemented with 10% fetal bovine serum andnon-essential amino acids. The virus infection and selection wereessentially as described previously (Ferbeyre et al., 2000). Afterone-week selection, the cells were either frozen for stock orimmediately used for further analysis. About 500,000 MEF cells wereplated on a 10-cm dish 24 hr before treatment. The cells were thenexposed to etoposide (20 μm) for 12 hr. After treatment, the cells werewashed with PBS and fed with normal medium. Another 36 hrs later, thecells were stained with PI and analyzed by flow cytometric analysis forapoptotic cells (SubG1) according to DNA content. In case of theFas-mediated apoptosis assay, the cells were treated with actinomycin D(0.25 μg/ml) and Fas antibody (100 ng/ml) as previously described (DiCristofano et al., 1999). In the case of oxidative stress response, theIMR-90 cells were treated with H₂O₂ (50 to 200 μM) for 24 hrs.

Detecting Acetylation Levels of p53 in Cells

The cells (human lung carcinoma cell lines H460 (wild-type p53) andH1299 (p53-null), human colon carcinoma HCT116 (wild-type p53), mouseembryonal carcinoma cell line F9 (wild-type p53), mouse embryonicfibroblast MEFs or others) were maintained in DMEM medium supplementedwith 10% fetal bovine serum. For DNA damage response, about 1 millioncells were plated on a 10-cm dish 24 hr before treatment. The cells werethen exposed to etoposide (20 μM) and or other drugs (0.5 μM of TSA, 5mM of nicotinamide, and 50 μM of ALLN) as indicated for 6 hr. Aftertreatment, the cells were harvested for western blot analysis. Therabbit polyclonal antibody specific for p300-mediated acetylatedp53-[α-p53(Ac)-C] was raised and purified against the acetylated humanp53 C-terminal peptide [p53 (Ac)-C: H-S55GQSTSRH55LMF-OH (5=acetylatedLysine)] as described before (Luo et al., 2000). In the case ofcotransfection assays testing for p53 acetylation levels, H1299 cellswere transfected with 5 μg of CMV-p53 plasmid DNA, 5 μg of CMV-p300plasmid DNA, and 10 μg of pcDNA3—Sir2α plasmid DNA as indicated. 24 hrafter the transfection, the cells were lysed in a Flag-lysis buffer (50mM Tris, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100 and 0.2%Sarkosyl, 1 mM DTT, 10% glycerol, pH 7.8) with fresh proteinaseinhibitors, 10 μM TSA and 5 mM nicotinamide (Sigma). The cell extractswere resolved by either 8% or 4-20% SDS-PAGE gels (Novex) and analyzedby western blot with α-p53(Ac)-C and α-p53(DO-1).

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1. A method for treating a subject afflicted with cancer comprisingadministering to the subject a therapeutically effective amount of anagent that inhibits the ability of Sir2α to inhibit p53-dependentapoptosis, thereby treating the subject.
 2. (canceled)
 3. The method ofclaim 1, wherein the subject is a human. 4-5. (canceled)
 6. The methodof claim 1, further comprising administering to the subject an agentthat enhances p53-dependent apoptosis via a mechanism other than Sir2αinhibition. 7-15. (canceled)
 16. A method for decreasing the amount ofdamage to a subject's cells caused by physical stress comprisingadministering to the subject a prophylactically effective amount of anagent that increases the amount of Sir2α in the subject's cells and/orthe ability of Sir2α to inhibit p53-dependent apoptosis in the subject'scells, thereby decreasing the amount of damage to the subject's cells.17. The method of claim 16, wherein the damage is selected from a groupconsisting of DNA damage and membrane damage.
 18. The method of claim16, wherein the physical stress is selected from the group consisting ofultraviolet radiation and oxidation.
 19. The method of claim 16, whereinthe agent is administered prior to and/or concurrently with theoccurrence of the physical stress.
 20. The method of claim 16, whereinthe agent is administered subsequent to the occurrence of physicalstress. 21-23. (canceled)
 24. A method for decreasing the amount ofdamage to a cell caused by physical stress comprising contacting thecell with an agent that increases the amount of Sir2α in the cell and/orthe ability of Sir2α to inhibit p53-dependent apoptosis in the cell,thereby decreasing the amount of damage to the cell.
 25. The method ofclaim 24, wherein the damage is selected from the group consisting ofDNA damage and membrane damage.
 26. The method of claim 24, wherein thephysical stress is selected from the group consisting of ultravioletradiation and oxidation.
 27. The method of claim 24, wherein the cell iscontacted with the agent prior to and/or concurrently with theoccurrence of the physical stress.
 28. The method of claim 24, whereinthe cell is contacted with an agent subsequent to the occurrences of thephysical stress.
 29. (canceled)
 30. The method of claim 24, wherein thecell is a human cell.
 31. The method of claim 24, wherein the agent is aSir2α-encoding nucleic acid. 32-33. (canceled)